CN116715709B

CN116715709B - Compound hydrochloride crystal form and application thereof

Info

Publication number: CN116715709B
Application number: CN202310969303.3A
Authority: CN
Inventors: 杨宏勃; 张安源; 张楠; 郑鹏飞; 曾勇; 王斌; 罗丽莲
Original assignee: Shaanxi Microbe Biotechnology Co ltd; China Grand Enterprise Inc
Current assignee: Shaanxi Microbe Biotechnology Co ltd; Yuanda Industry Holding Co ltd
Priority date: 2023-08-03
Filing date: 2023-08-03
Publication date: 2023-10-27
Anticipated expiration: 2043-08-03
Also published as: CN116715709A; CN117645640A

Abstract

The application relates to a crystal form I of an agricultural antibiotic 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-yl amino-alpha-iminoacetate and an amorphous substance thereof, wherein the crystal form I shows good stability under high-temperature high-humidity illumination environment, can better meet the requirements of pesticides in production processing and transportation storage, shows excellent effects in preparation processing and preparation stability, and can better meet the requirements of mixed preparation processing and stable shelf life. Meanwhile, the amorphous form of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt exhibits unexpected effects in terms of plant disease control effect.

Description

Compound hydrochloride crystal form and application thereof

Technical Field

The application belongs to the technical field of agriculture, and particularly relates to a crystal form and an amorphous substance, an original drug, a mother drug or a preparation of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-amino-alpha-iminoacetate and application thereof.

Background

5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetic acid (CAS 6980-18-3) belongs to an aminoglycoside type antibiotic, and since the 20 th century 60-70 s 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetic acid has been found to date, which has been widely used for the control of various diseases of various crops including rice, potato, cabbage, melon and the like. Because of the excellent control effect and the environment-friendly characteristic, the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino-alpha-iminoacetic acid is a main biopesticide product for controlling crop diseases at present. 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetic acid is extremely unstable in a slightly alkaline environment, and 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetic acid is a hygroscopic compound, and a more stable, less hygroscopic 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetic acid product is desired by production and processing enterprises from the viewpoints of transportation, preservation, quality control and the like.

At the beginning of the discovery of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetic acid, ikekawa T et al briefly examined the crystalline form of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetic acid hydrobromide (The Journal of Antibiotics, 01 Jan 1966, 19 (1): 49-50). CN115925475a reports the use of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetic acid phosphate for the purpose of supplementing phosphorus elements while controlling pathogens. CN108822167a reports 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salts and their crystals in the form of white needles or flakes. CN106083951B and CN109666051B report the extraction of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt by solvent elution crystallization with addition of acetone, methanol, ethanol, etc. in water.

Although the prior art has disclosed processes for preparing highly pure 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salts, the crystalline products obtained by different crystallization processes tend to differ in terms of their physicochemical properties and in terms of formulation processing properties. Thus, a stable and formulation processing and storage friendly crystalline product of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetate is a sought after goal for biopesticide enterprises.

Disclosure of Invention

The present application provides a solution to the above problems existing in the prior art.

According to one aspect of the present application there is provided crystalline form I of the agricultural antibiotic 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt, wherein form I uses Cu-ka radiation and the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2Θ values ± 0.2 ° comprise 8.66, 10.11, 11.05 and 13.3.

Alternatively, form I uses Cu-ka radiation and the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2θ values ± 0.2 ° further comprise one or both of 13.92 and 15.56, preferably further comprising one or both of 16.47 and 17.29.

Alternatively, form I particle size D ₉₀ From 10 μm to 200. Mu.m, preferably from 15 μm to 150. Mu.m, more preferably from 20 μm to 120. Mu.m.

Alternatively, form I is granular or rod-shaped.

Differential scanning calorimetric analysis (DSC) shows that the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate hydrochloride form I has an endothermic signal that persists over an interval from 110±2 ℃ to 210±2 ℃ and an endothermic signal at 226±2 ℃; compared with the heat absorption peak height (or peak valley), the crystal form I has a strong heat absorption process at 226+/-2 ℃ and has a weak heat absorption process at a range of 110+/-2 ℃ to 210+/-2 ℃.

Thermogravimetric analysis (TGA) shows that the crystal form I has substantially less than 5% weight loss during heating to 220±2 ℃ at 100±2 ℃ and substantially less than 15.5% weight loss during heating to 255±2 ℃ at 220±2 ℃.

According to another aspect of the present application there is provided an amorphous form of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate.

Alternatively, the amorphous material has an X-ray powder diffraction peak as shown in fig. 6.

DSC shows that the amorphous object has heat absorption signals at 73+/-2 ℃ and 187+/-2 ℃;

the TGA results show that the amorphous material has substantially less than 9% weight loss during heating to 175±2 ℃ and less than 15% weight loss during heating to 175±2 ℃ to 255±2 ℃.

Optionally, the amorphous form of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt is of lamellar structure.

Alternatively, the amorphous D ₉₀ From 10 μm to 200. Mu.m, preferably from 15 μm to 150. Mu.m, more preferably from 20 μm to 120. Mu.m.

According to another aspect of the present application there is provided a prodrug or a parent drug comprising the crystalline form I and/or amorphous form as described above.

The content of the crystal form I and/or the amorphous substance in the crude drug is not less than 65%, such as not less than 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80%, based on the total mass; preferably, not less than 85% or 90% or 95%.

The content of form I and/or amorphous in the parent drug is not less than 5% or 10%, such as not less than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80% by total mass; preferably not less than 85% or 90% or 95%.

According to another aspect of the present application there is provided a formulation or pesticidal composition comprising the crystalline form I and/or amorphous form as described above and an adjuvant.

Optionally, the weight percentage of the crystalline form I and/or amorphous form in the formulation or pesticide composition is at least 0.001%.

Optionally, the formulation is selected from any one of powder, granule, large granule, fine granule, microparticle, microcapsule, wettable powder, oil dispersion powder, water dispersion granule, emulsion, effervescent granule, dispersible tablet, effervescent tablet, sustained release agent, sustained release block, sustained release tube, sustained release granule, soluble powder, soluble granule, soluble tablet, soluble liquid, aqueous solution, soluble solution, oil, film spreading oil, ultra low volume liquid, ultra low volume microcapsule suspension, emulsifiable, latex, dispersible liquid, paste, concentrated gelatin, aqueous emulsion, oil emulsion, microemulsion, grease, suspension, microcapsule suspension, oil suspension, suspoemulsion, seed treatment dispersible powder, seed treatment soluble powder, seed treatment liquid, seed treatment emulsion, seed treatment suspension, seed treatment microcapsule suspension. Preferably, the crystalline form I or amorphous solid according to the present application exhibits excellent workability and solid application controlling effect in a pesticidal solid preparation. Specifically, the crystal form I or the amorphous substance is used for wettable powder or powder; wherein the particle size D of form I and/or of the amorphous form in the powder or wettable powder ₉₀ A preferred particle size D of 5 μm to 80. Mu.m ₉₀ From 10 μm to 50 μm, or from 15 μm to 45 μm, or from 20 μm, 30 μm, 40 μm, 60 μm and 70 μm.

According to another aspect of the present application there is provided the use of the above-described crystalline form I, the above-described amorphous form, the above-described crude drug or the above-described parent drug, or the above-described formulation or pesticide composition in a plant disease control process.

According to another aspect of the present application, there is provided the use of the above-mentioned crystalline form I, the above-mentioned amorphous form, the above-mentioned crude drug or the above-mentioned parent drug, or the above-mentioned preparation or pesticide composition for the preparation of a plant disease control preparation.

The application has the positive progress effects that:

the applicant believes that the crystal form I provided by the application has good stability and almost no hygroscopicity, can be kept stable under all conditions of illumination, high temperature, high humidity and acceleration, effectively prolongs the shelf life of medicine storage, and can better meet the requirements of pesticides in production, processing, transportation and storage. Meanwhile, the crystal form I also has good technical effect on the prevention and treatment of plant diseases. Unexpectedly, the amorphous material shows the optimal effect in the aspect of plant disease control effect, and the application effect of the agricultural terminal is more outstanding.

Drawings

FIG. 1 is a diagram of a compound form I hydrogen nuclear magnetic analysis (1H-NMR);

FIG. 2 is an X-ray powder diffraction (XRPD) pattern for form I, with 2 θ (o) on the abscissa and intensity (counts) on the ordinate;

FIG. 3 is a diagram of a compound form II hydrogen nuclear magnetic analysis (1H-NMR);

FIG. 4 is an X-ray powder diffraction (XRPD) pattern for form II, with 2 theta (o) on the abscissa and intensity (counts) on the ordinate;

FIG. 5 is a chart of hydrogen nuclear magnetic analysis (1H-NMR) of an amorphous material;

FIG. 6 is an amorphous X-ray powder diffraction (XRPD) pattern with 2 theta (o) on the abscissa and intensity (counts) on the ordinate;

FIG. 7 is a differential scanning calorimeter analysis (DSC) and thermogravimetric analysis (TGA) profile of form I;

FIG. 8 is a differential scanning calorimeter analysis (DSC) and thermogravimetric analysis (TGA) profile of form II;

FIG. 9 is a differential scanning calorimeter analysis (DSC) and thermogravimetric analysis (TGA) profile of an amorphous form;

FIG. 10 is a polarization microscopic analysis (PLM) spectrum of form I;

FIG. 11 is a polarized microscope analysis (PLM) image of form II;

FIG. 12 is a Polarized Light Microscopy (PLM) image of an amorphous;

FIG. 13 is a form I particle size distribution measurement (PSD) image;

FIG. 14 is a form II particle size distribution measurement (PSD) image;

FIG. 15 is a dynamic moisture desorption assay (DVS) profile of form I;

FIG. 16 is a stability measurement of form I;

FIG. 17 shows the results of a potted antimicrobial test, wherein a-1 is a front view of a blank control blade, a-2 is a back side of the blank control blade, b-1 is a front view of a crystal form II powder test blade, b-2 is a back side view of the crystal form II powder test blade, c-1 is a front view of a crystal form I powder test blade, c-2 is a back side view of the crystal form I powder test blade, d-1 is a front view of an amorphous powder test blade, and d-2 is a back side view of an amorphous powder test blade.

Detailed Description

The term "solvates" refers to those forms of the crystalline forms of the compounds of the present application that are used to form complexes with molecules of solvents (e.g., water, organic solvents such as formic acid, toluene, etc.) by coordination. Hydrates are a specific form of solvate in which coordination with water occurs. For example, the solvate may be a hydrate.

The term "crude drug" refers to a product obtained during the production process and composed of an active ingredient and related impurities, and small amounts of additives may be added if necessary.

The term "parent drug" refers to a product obtained during the production process, consisting of the active principle and related impurities, possibly with small amounts of necessary additives and suitable diluents.

The term "formulation" refers to a stable product processed from a pesticide base stock (parent) and suitable adjuvants, or processed by methods such as biological fermentation, plant extraction, etc.

The term "adjuvant" refers to any substance, other than the active ingredient, which is added to a pesticidal product, does not itself have pesticidal activity and active ingredient function, but is capable of or contributes to the enhancement, improvement, or physical and chemical properties of the pesticidal product, of a single component or of multiple components.

The term "plant disease" refers to a phenomenon that a plant is significantly hindered from growing and developing due to infection by other organisms and adverse abiotic factors during the growth and development of the plant, and pathological changes and even death occur in physiological and tissue structures inside and outside the plant, resulting in reduced yield and deteriorated quality. "plant diseases" according to the application refer in particular to diseases of plants caused by infestation by other organisms including, but not limited to, fungi (e.g. cucumber downy mildew caused by downy mildew of the species downy mildew of the genus copaiba), or bacteria (e.g. bacterial angular leaf spot of melons) and the like.

The terms "about", "about" and "substantially" refer to a numerical variation in the content of the error range of a normal experiment or measurement, e.g., that "substantially" represents an error of no more than 15%, preferably no more than 10%.

In one embodiment, the crystalline form I of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt of the present application is a hydrate.

In a specific embodiment, the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetate hydrochloride form I of the present application uses Cu-ka radiation, and the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2θ values ± 0.2 ° include 8.66, 10.11, 11.05 and 13.3, preferably, form I uses Cu-ka radiation, and the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2θ values ± 0.2 ° include 8.66, 10.11, 11.05, 13.3, 13.92 and 15.56; preferably, the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2θ values±0.2° include 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47 and 17.29; more preferably, the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2θ values±0.2° include 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47, 17.29, 18.59, 19.25, or 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47, 17.29, 18.59, 19.25, 20.41 and 20.78.

In a specific embodiment, the compound 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino-alphaForm I of the iminoacetate is granular or rod-shaped, and the grain diameter D of the form I meets the requirements of actual production, convenience in solid-liquid filtration and convenience in subsequent processing of pesticide solid preparations ₉₀ 10 μm to 200 μm. Preferably, form I particle size D ₉₀ 15 μm to 150 μm, 20 μm to 120 μm, e.g. form I particle size D ₉₀ But may also be 10, 15, 25, 35, 45, 55, 65, 75, 85, 95, 100, 110, 130, 140, 150, 160, 170, 180, 190 or 200 μm.

In a specific embodiment, differential scanning calorimetric analysis (DSC) shows that the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate form I has a continuous endothermic signal in the interval from 110±2 ℃ to 210±2 ℃ and an endothermic signal at 226±2 ℃; compared with the heat absorption peak height (or peak valley), the crystal form I has a strong heat absorption process at 226+/-2 ℃ and has a weak heat absorption process at a range of 110+/-2 ℃ to 210+/-2 ℃.

In a specific embodiment, the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetate salt prepared according to the present application is amorphous and has an XRPD diffraction peak pattern as shown in FIG. 6.

DSC shows that the amorphous substance has endothermic signals at 73+/-2 ℃ and 187+/-2 ℃.

TGA shows that the amorphous mass has substantially less than 9% weight loss during heating to 175±2 ℃ and substantially less than 15% weight loss during heating to 175±2 ℃ to 255±2 ℃.

Alternatively, the amorphous particle diameter D ₉₀ 10 μm to 200 μm. Preferably, form I particle size D ₉₀ 15 μm to 150 μm, 20 μm to 120 μm, e.g. form I particlesDiameter D ₉₀ But may also be 10, 15, 25, 35, 45, 55, 65, 75, 85, 95, 100, 110, 130, 140, 150, 160, 170, 180, 190 or 200 μm.

In one embodiment, the crystalline solid may be prepared by a solvent-out anti-drip method, a liquid gas phase diffusion method, a single solvent room temperature suspension method, a single solvent Gao Wenxuan float method, a binary solvent positive drip method, a single solvent cooling method, a binary solvent cooling method, or the like.

Preferably, the crystal form I is prepared by a solution precipitation anti-dripping method, a liquid gas phase diffusion method and the like, is not limited by the examples of the preparation of the application, and is prepared by other crystallization methods such as a single solvent room temperature suspension method, a single solvent Gao Wenxuan float method, a binary solvent normal dripping method, a single solvent cooling method, a binary solvent cooling method and the like, and the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino-alpha-iminoacetate crystal form I also falls into the protection scope of the application.

Preferably, the amorphous material is prepared by freeze-drying, which may be selected from the group consisting of cyclic freeze-drying or medium freeze-drying (e.g. dry ice freeze-drying or nitrogen freeze-drying).

Preferably, the solvency anti-drip method comprises the steps of: the aqueous solution containing 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino-alpha-iminoacetate is dripped into a poor solvent, and suspended for 10 min-120 min after precipitation, and filtered to obtain a solid. The dripping process is optionally stirred or not stirred, and the stirring speed is 20 rpm-120rpm; the poor solvent is dioxane or ethylene glycol dimethyl ether.

Preferably, the liquid gas phase diffusion process comprises the steps of: weighing a certain amount of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino-alpha-iminoacetate salt solution dissolved in a good solvent, placing the clear solution in a poor solvent atmosphere, standing at room temperature until a solid is precipitated, and filtering. The good solvent is water or formamide, and the poor solvent is trifluoroethanol.

In a specific embodiment, the application provides a crude drug or a mother drug containing the crystal form I or the amorphous substance according to the application, wherein the content of the crystal form I in the crude drug is not less than 65%, such as not less than 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80% by total mass; preferably, not less than 85% or 90% or 95%.

In a specific embodiment, the present application provides a formulation or pesticidal composition comprising form I and/or an amorphous form according to the present application, preferably at least 0.001% by weight of the form I and/or amorphous form in the formulation, e.g. at least 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% by weight of the form I and/or amorphous form in the formulation. Preferably, the weight percentage of the crystalline form I and/or amorphous form in the formulation is 0.1% -10%, 0.5-7%, or 1-5%.

In another embodiment, there is provided the use of the above-described crystalline form I, the above-described amorphous form, the above-described crude drug or parent drug, or the above-described formulation or pesticide composition for the preparation of a plant disease controlling medicament.

Specific examples:

all commercial reagents and solvents were not further purified.

Sample detection in the examples was performed as follows.

1. X-ray powder diffraction (XRPD)

The solid samples obtained from the experiments were analyzed by means of an X-ray powder diffractometer Bruker D8 Advance (Bruker, GER). The 2 theta scanning angle is from 3 degrees to 45 degrees, the scanning step length is 0.02 degrees, and the exposure time is 0.08 seconds. The test method is Cu target K alpha 1 rays, voltage 40 kV and current 40 mA, and the sample disk is a zero background sample disk.

2. Differential scanning calorimetric analysis (DSC)

The differential scanning calorimeter was model TA Discovery 2500 (TA, US). 1-2 mg samples are accurately weighed and placed in a pricked DSC Tzero sample tray, heated to a final temperature at a rate of 10 ℃ per minute, and purged with nitrogen in a furnace at a rate of 50 mL per minute.

3. Thermogravimetric analysis (TGA)

The thermogravimetric analyzer was model TA Discovery 55 (TA, US). Samples 2-5 mg were placed in equilibrated open aluminum sample trays and automatically weighed in a TGA furnace. The sample was heated to the final temperature at a rate of 10 ℃ per minute with a nitrogen purge rate of 60 mL per minute at the sample and 40 mL per minute at the balance.

4. Nuclear magnetic analysis (1H NMR)

Several milligrams of solid sample were dissolved in dimethyl sulfoxide-d 6 solvent and subjected to nuclear magnetic analysis on Bruker AVANCE NEO (Bruker, GER).

5. Polarized light microscopic analysis (PLM)

The polarizing microscope was Nikon Ci-POL (Nikon, JP). A small amount of sample is placed on a glass slide, and a proper lens is selected to observe the appearance of the sample.

6. Particle size distribution measurement (PSD)

The laser particle size analyzer model was Mastersizer 3000 (Malvern Panalytical, UK). A 20 mg sample was taken and dispersed in 8 mL dispersant, and the sample dispersion unit was added until the opacity was 10-20% and the stirring speed was 2000 rpm, duration 10s, the dispersant was ethanol, the scattering model was Mie, and the analytical model was universal.

7. Dynamic moisture desorption analysis (DVS)

Dynamic moisture sorption and desorption analysis was performed using DVS Intrinsic (SMS, UK). The test adopts a gradient mode, the humidity change is 50% -95% -0% -50%, the humidity change amount of each gradient is 10% in the range of 0% -90%, the gradient end point is judged in a dm/dt mode, the dm/dt is less than 0.002% and is maintained for 10 minutes as the gradient end point, or each gradient is maintained for 180 minutes at maximum. After the test is completed, XRPD analysis is performed on the sample to confirm whether the solid morphology is changed.

8. Stability study

(1) Influence factor experiment

The 20 mg samples were weighed into weighing flasks, placed open at high temperature (60 ℃) and high humidity (25 ℃ C./92.5% RH) or under light (25 ℃ C./4500 Lux), sampled for 7 days and 15 days, observed for appearance, subjected to XRPD characterization and High Performance Liquid Chromatography (HPLC) testing, and compared to the results for 0 day.

(2) Accelerated stability test

The 20 mg samples were weighed into weighing flasks, left open under acceleration (40 ℃ C./75% RH), sampled for 7 and 15 days, observed for appearance, XRPD characterization and HPLC testing, and compared to the results for 0 days.

9、HPLC

The model of high performance liquid chromatography is ACQUITY ARC-2489 (Waters, US);

mobile phase: acetonitrile, sodium dodecyl sulfonate solution = 1:4, v/v (phosphoric acid pH adjusted to 2.5), sodium dodecyl sulfonate solution formulation: 0.8 g sodium dodecyl sulfate solid was dissolved in 400 ml ultrapure water;

chromatographic column: CORTECS C18 4.6X10 150 mm, 2.7 μm

Detection wavelength: 210nm of

Flow rate: 1.2mL/min

Column temperature: 30 DEG C

Sample injection amount: 5 mu L

Preparation example (1)

Comparative example 1: preparation of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt form II

Providing a fermentation broth comprising 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetic acid, specifically: streptomyces parvulus is adopted as a producing strain, and fermentation broth is obtained through multistage fermentation in a culture medium containing raw materials such as low-temperature soybean cake powder, soybean oil, yeast powder, liquid sugar and the like. According to the purification and crystallization method described in the embodiment of CN106083951B, through pretreatment of fermentation broth, filtration by ceramic membrane, decolorization by macroporous resin, nanofiltration concentration and other steps, the crystallization process is respectively added with organic solvents of acetone, methanol, ethanol, propanol or isopropanol for crystallization, and 5 solid crystallization products are prepared;

the solid crystalline product was dried in vacuo and then subjected to NMR and XRPD measurements, and NMR showed that the solid crystalline product was 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino-. Alpha. -iminoacetate salt, wherein the NMR spectrum of the solid crystalline product obtained by methanol crystallization as an organic solvent was shown in FIG. 3. XRPD results show that the above solid crystalline product is in one form, referred to as form II, wherein the XRPD pattern of the solid crystalline product crystallized from the organic solvent acetone is shown in fig. 4.

Comparative example 2: preparation of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt form II

Streptomyces parvulus is adopted as a producing strain, and fermentation broth is obtained through multistage fermentation in a culture medium containing raw materials such as low-temperature soybean cake powder, soybean oil, yeast powder, liquid sugar and the like. Purifying and crystallizing according to the method described in the embodiment of CN109666051B, wherein the crystallization mode adopts a cooling crystallization mode or an organic solvent acetone or ethanol crystallization mode, and 3 solid crystallization products are respectively prepared; the solid crystalline product was dried under vacuum at room temperature and then subjected to NMR and XRPD measurements, respectively, and NMR showed that the solid crystalline product was 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate and XRPD showed that the solid crystalline product was the same as the crystalline form obtained in comparative example 1, and was crystalline form II.

Example 1: preparation of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt form I

a) Acidifying the fermentation broth containing 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino-alpha-iminoacetic acid with oxalic acid, and filtering;

b) Collecting filtrate, adsorbing with strong acid cation exchange resin, and resolving;

c) Concentrating the ammonium chloride analysis solution through a nanofiltration membrane, and further decoloring through active carbon;

d) Filtering the decolorized solution, and vacuum concentrating to obtain vacuum concentrated solution;

e) Taking the vacuum concentrated solution prepared in the step d of 10 ml, adding the concentrated solution into dioxane (poor solvent) with the volume of 20 times at room temperature, suspending for 30-40min at the stirring speed of 60 rpm, and filtering to obtain a precipitate;

f) Drying the obtained precipitate at room temperature to obtain a high-purity solid crystal product, and carrying out NMR and XRPD analysis on the solid crystal product respectively; the NMR of the solid crystalline product was analyzed as shown in figure 1, indicating that it was 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate, XRPD as shown in figure 2, referred to as form I.

Example 2: preparation of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt form I

A high purity solid crystalline product was prepared following steps a-f of example 1, except that the poor solvent was replaced with ethylene glycol dimethyl ether, which was identified by NMR and XRPD as 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate form I.

Example 3: preparation of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt form I

a) Taking 250 mg the dried solid crystalline product of example 1;

b) After dissolving the solid crystalline product in 4 ml water;

c) Taking the solution in the step b of 1 ml, placing the concentrated solution in the atmosphere of trifluoroethanol, and standing at room temperature until solid is separated out;

d) The solution in the system with solid precipitated was removed by syringe and NMR and XRPD tests were performed on the solid sample, which showed that the solid sample was crystalline form I of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt.

Example 4: preparation of amorphous 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt

a) Taking 100 mg of the dried solid crystalline product of example 1;

b) Dissolving the solid crystalline product in 2ml water;

c) Freezing the solution in the step b by using dry ice, and freeze-drying the solution in a freeze dryer for 1 day;

d) The freeze-dried material was subjected to NMR and XRPD measurements, and the NMR result showed that it was 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate (as shown in fig. 5), and the XRPD result showed that it was an amorphous material without distinct characteristic peaks (as shown in fig. 6).

Example 5: preparation of powders

About 70g of three dry solids of form II, form I and amorphous form were prepared according to the methods of comparative example 1, example 1 and example 4, at 98.1%, 98.7% and 96.3%, respectively, for use as a drug substance/parent drug of the formulation.

The raw materials/mother medicines are respectively crushed by adopting a small vertical sand mill and zirconium beads (phi 1.0-1.2 mm, shanghai) new material technology Co., ltd.), wherein the using amount of the zirconium beads is 40 g, the rotating speed of the sand mill is 1500 r/min, the grinding time is 30 min, and the zirconium beads and the fine powder are separated after the grinding is finished.

PSD analysis showed that the particle size D90 of the three crude drug/parent drug fines was 36.6 μm-40.2 μm.

4 g g of each of the three raw materials/mother materials is taken out from the fine powder, and the three fine powders and 96g diatomite are respectively screened by 200 meshes and then fully mixed to prepare three powders.

Example 6: preparation of wettable powder

The amounts of the components of the wettable powder are shown in the following table:

the wettable powder is prepared by the following steps:

1) Taking the fine powder of the crude drugs/the mother drugs of the crystal form I and the amorphous substance prepared in the example 5 respectively according to the table above for standby;

2) Fully mixing copper, sodium dodecyl sulfate, sodium lignin sulfonate, white carbon black and diatomite, and crushing by a superfine crusher to obtain auxiliary agent powder;

3) And (3) fully mixing the crude drug/mother drug fine powder with the auxiliary agent powder prepared in the step (2) to obtain the wettable powder.

Basic characterization of the (di) crystalline form

(1) 1H NMR analysis

NMR showed that form I (shown in FIG. 1), form II (shown in FIG. 3) and the amorphous form (shown in FIG. 5) were all 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt compounds.

(2) XRPD diffraction peak data analysis

XRPD diffraction peak data of the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt form I of the compound of the application are shown in table 1; XRPD diffraction peak data for form II are shown in table 2.

TABLE 1 XRPD diffraction peak data for form I

As can be seen from table 1, form I uses Cu-ka radiation, and the main characteristic diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2θ values ± 0.2 ° include any one or more of the characteristic diffraction peaks of 8.66, 10.11, 11.05 and 13.3, and further include any one or more of 13.92, 15.56, 16.47 and 17.29, specifically including 8.66, 10.11, 11.05, 13.3, 13.92 and 15.56; or 8.66, 10.11, 11.05, 13.3, 16.47 and 17.29; or 8.66, 10.11, 11.05, 13.3, 13.92, 15.56, 16.47 and 17.29.

TABLE 2 XRPD diffraction peak data for form II

As can be seen from table 2, the main characteristic diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2θ values ± 0.2 ° using Cu-ka radiation for form II include 8.91, 10.03, 10.51, 12.12, 15.01, 16.40, 17.14 and 26.68; the characteristic diffraction peaks of the crystal form II do not contain characteristic diffraction peaks such as 8.66, 11.05 and 13.30, but contain characteristic diffraction peaks such as 8.91, 10.51 and 12.12, compared with the crystal form I.

As can be seen from fig. 6, an amorphous material having no significant diffraction peak was prepared by the freeze-drying method.

In summary, three solid products, form I, form II and amorphous, were prepared by the preparation method of the present application, and by comparing the XRPD patterns and diffraction peak data of form I and form II, both of which are different forms.

(3) DSC and TGA analysis

The DSC spectrum and TGA spectrum of the crystal form I are shown in figure 7; the DSC profile and TGA profile of form II are shown in FIG. 8.

DSC shows that the crystal form I has a continuous heat absorption signal in a range from 110+/-2 ℃ to 210+/-2 ℃ and has a heat absorption signal at 226+/-2 ℃; compared with the heat absorption peak height (or peak valley), the crystal form I has a strong heat absorption process at 226+/-2 ℃ and a weak heat absorption process in the interval.

TGA shows that the crystal form I has about 0.2% weight loss during heating to 100±2 ℃, substantially less than 5% weight loss during heating to 100±2 ℃ to 220±2 ℃, and substantially less than 15.5% weight loss during heating to 255±2 ℃.

DSC shows that the crystal form II has a weak heat absorption signal at 147+/-2 ℃ and has a strong heat absorption signal at 215+/-2 ℃; compared with the heat absorption peak height (or peak valley), the crystal form I is a strong heat absorption process at 215+/-2 ℃ and is a weak heat absorption process at 147+/-2 ℃.

TGA shows that the crystal form II has substantially less than 6% weight loss during heating to 195±2 ℃ and substantially less than 15% weight loss during heating to 250±2 ℃ at 220±2 ℃.

DSC shows that the amorphous substance has endothermic signals at 73+/-2 ℃ and 187+/-2 ℃;

the TGA results show that the amorphous form has less than 9% weight loss during heating to 175±2 ℃, and substantially less than 15% weight loss during heating to 255±2 ℃.

(4) PLM and PSD analysis

PLM images show that form I is predominantly granular (as shown in fig. 10); particle size distribution analysis shows that the crystal form I has particle size D ₅₀ 64.2 μm, D ₉₀ 119 μm (as shown in FIG. 13).

PLM images show that form II is predominantly acicular (as shown in fig. 11); particle size distribution analysis shows that the particle size D of the crystal form II ₅₀ 24.7 μm, D ₉₀ 70.1 μm (as shown in FIG. 14).

PLM images showed that the amorphous material was in the form of flakes (as shown in FIG. 12), particle size D ₅₀ 30.2 μm.

(III) powder flowability

To verify the processing properties of the different solid products, the flowability of the three fine powders prepared in example 5 was tested according to the "angle of repose" method in the powder flowability determination guidelines issued by the national formulary committee, year 12, month 19. Wherein, the test is carried out by adopting a fixed funnel height mode (10 cm), the test temperature is room temperature, the humidity is about 5%, the test fine powder consumption is 60g, and the test result is shown in table 3.

TABLE 3 evaluation of powder flowability

As can be seen from table 3, the crushed crystalline form I and amorphous form have better powder flowability than crystalline form II, indicating that the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetate salt of crystalline form I and amorphous form is more advantageous for the subsequent processing and utilization of the compound and formulation, wherein the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino- α -iminoacetate salt of crystalline form I structure shows the best effect in terms of processing, and the hygroscopicity and stability of crystalline form I, crystalline form II and amorphous form will be further examined later.

(IV) DVS analysis

As shown in fig. 15, form I only increased by 1.57% at 95% humidity, 0.40% at 80% humidity, and 1.02% at 0% humidity, indicating that form I was substantially free of hygroscopicity, indicating that form I was also a stable solid product in a high humidity environment.

In contrast, the crystal form II has 2.2% of moisture absorption and weight gain under the high humidity environment of 95%, 0.74% of weight gain under the humidity of 80%, and shows weak moisture absorption under the high humidity environment; the amorphous material exhibits the general hygroscopic characteristics of the amorphous material.

(fifth) stability determination

Form I and form II were subjected to stability studies at high temperature (60 ℃), high humidity (25 ℃ per 92.5% RH), light (25 ℃ per 4500 Lux) and acceleration (40 ℃ per 75% RH), sampled at 7 days and 15 days, respectively, observed for appearance, XRPD characterization and HPLC testing, and compared to the results as received. The results show that the crystal form I is stable under the conditions of high temperature, high humidity, illumination and acceleration stability, and is specifically expressed as follows: the appearance was unchanged, no crystal form transformation occurred, and the chemical purity was not significantly changed (as shown in table 4, fig. 16), indicating that form I is a stable solid crystalline product.

The stability result of the crystal form II shows that the crystal form II keeps stable appearance under the high temperature and high humidity test condition for 15 days, the appearance changes from white to yellow after 7 days of illumination test, and the color changes to light brown after 15 days, which indicates that the crystal form II is unstable under continuous illumination.

TABLE 4 results of experiments on different environmental impact factors for form I

Sixth, potted plant antibacterial effect

1. Preparation of cucumber downy mildew spore suspension

The cucumber downy mildew is picked from the leaves naturally occurring in a demonstration park of the ecological agriculture of the Wei-first, the collected leaves are used for removing the mildew dirt on the back surfaces of leaf spots by a writing brush dipped with distilled water, the leaves are placed in a climatic chamber with the temperature of 25 ℃ and the relative humidity of 80% for dark culture of 16 h, so that fresh sporangia are produced; brushing fresh sporangium into culture dish containing distilled water with brush pen, filtering twice to obtain spore suspension (concentration of 2×10) ⁵ -4×10 ⁵ and/mL), ready for use.

2. Cucumber indoor planting and culturing

Cucumber plants (Xinong No. 58, 20 pots in total, 1 plant/pot) were grown using plastic pots with a diameter of 5 cm, and cultured to a plant height of about 1 meter under conditions of a temperature of 20 ℃ -28 ℃ (day and night), a relative humidity of (90+ -5)%, and a fluorescent lamp of 12 h, and 12 cucumber seedlings of which growth vigor is equivalent were selected as subsequent experimental samples.

3. Preventive foliage application

Two leaves with equivalent height (calculated from soil surface) and leaf width of about 5-8 cm are selected from each of 12 cucumber seedlings. The three mixed powders prepared in the example 5 are respectively filled in powder spraying bottles with the same specification, and preventive application is carried out according to the powder pesticide application mode: two injections of powder were performed at a height of about 40 and cm a from the leaf surface, each powder was sprayed with three, 6 leaves each as parallel experiments, and the blank group was sprayed with only the diatomaceous earth dry powder. Corresponding marks and records are made on the leaves (group A is blank group, group B is powder containing crystal form II, group C is powder containing crystal form I, group D is powder containing amorphous substance), and cucumber downy mildew inoculation is carried out after continuous culture for 48 h according to the previous planting conditions.

4. Cucumber downy mildew inoculation and therapeutic application

And (3) pouring the prepared spore suspension into a spray can, spraying the spore suspension onto the leaf surfaces twice from the height of about 20 to cm of the selected leaf surfaces, immediately sleeving a transparent plastic bag after the spraying is finished, and fastening a bag fastener, so that the downy mildew inoculation of 24 leaves on 12 cucumber seedlings is completed. After inoculation, the transparent plastic bags are taken down respectively at the positions of 2 h, 6 h and 10 h, the inoculated leaves are wetted with pure water by spraying, and then bagging treatment is continued. After the inoculation is completed 24 h, therapeutic application is carried out according to a preventive application mode (A group is sprayed with diatomite dry powder, B group is sprayed with powder containing crystal form II, C group is sprayed with powder containing crystal form I, D group is sprayed with powder containing amorphous matters), leaves are cleaned after 15 days of therapeutic application, experimental leaf surface conditions are observed and recorded, and statistical analysis is carried out.

The disease states are classified according to the areas of the disease spots, and the classification standards are as follows:

level 0: the leaf has no disease spots;

stage 1: the area of the disease spots accounts for less than 5% of the whole blade area;

3 stages: the area of the disease spots accounts for 6-10% of the whole leaf area;

5 stages: the area of the lesion accounts for 11-25% of the whole leaf area;

7 stages: the area of the disease spots accounts for 26-50% of the whole leaf area;

stage 9: the area of the disease spots accounts for more than 50% of the whole blade area;

disease index = { [ Σ (number of leaf of each stage of disease of each treatment×number of corresponding stage) ]/total leaf number of investigation×9} ×100;

control effect (%) = [ (blank control disease index-agent treatment disease index)/blank control disease index ] ×100;

the results of the four groups of experiments are shown in Table 5, and the partial effects are shown in FIG. 17.

TABLE 5 foliar control effect

From the aspects of plant disease index and control effect, it is surprising that the crystal form I, the crystal form II and the amorphous substance have remarkable differences in the plant disease control effect, wherein the plant disease control effect of the powder containing the amorphous substance is the best, and the powder containing the crystal form I is the next. The results show that the amorphous and the crystal form I of the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino-alpha-iminoacetate have better plant disease control effect than the crystal form II because of different crystal forms and amorphous matters of the 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxy) pyran-3-ylamino-alpha-iminoacetate and the like.

The foregoing embodiments have been provided for the purpose of illustrating the general principles of the present application, and are not meant to limit the scope of the application, but to limit the scope of the application.

Claims

1. An agricultural antibiotic 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt form I, characterized in that said form I uses Cu-ka radiation, and the characteristic diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2Θ values ± 0.2 ° include 8.66, 10.11, 11.05 and 13.3.

2. Form I according to claim 1, characterized in that it uses Cu-ka radiation, characterized diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2Θ values ± 0.2 ° further comprising 13.92 and 15.56.

3. Form I according to claim 2, characterized in that it uses Cu-ka radiation, characterized diffraction peaks of the X-ray powder diffraction pattern expressed in terms of 2Θ values ± 0.2 ° further comprising 16.47 and 17.29.

4. Form I according to any one of claims 1-3, characterized in that the particle size D of form I ₉₀ 10 μm to 200 μm.

5. Form I according to claim 4, characterized in that the particle size D of form I ₉₀ 15 μm to 150 μm.

6. A prodrug or parent drug, characterized in that it contains the crystalline form I according to any one of claims 1 to 5.

7. A formulation or pesticide composition, characterized in that it contains the crystalline form I according to any one of claims 1 to 5.

8. The formulation or pesticide composition of claim 7, wherein the weight percent of form I in the formulation or pesticide composition is at least 0.001%.

9. The formulation or pesticide composition of claim 7 or 8, wherein the formulation or pesticide composition is in a form selected from any one of a powder, granule, dispersible tablet, slow release, sol, oil, emulsion, dispersible, paste, gel concentrate, emulsion in water, emulsion in oil, microemulsion, grease, suspension, suspoemulsion, seed treatment emulsion, suspension seed coating.

10. The formulation or pesticide composition of claim 7 or 8, wherein the formulation or pesticide composition is in a dosage form selected from any one of wettable powder, oil dispersion powder, soluble powder, seed treatment dispersible powder, seed treatment soluble powder, macrogranules, fine granules, microgranules, micro-capsules, water dispersible granules, emulsion granules, effervescent granules, slow release granules, soluble granules, effervescent tablets, soluble tablets, slow release blocks, slow release tubes, soluble solutions, aqueous solutions, ultra low volume solutions, seed treatment solutions, film spreading oil, ultra low volume micro-capsule suspensions, oil suspensions, seed treatment suspensions or seed treatment micro-capsule suspensions.

11. The formulation or pesticide composition of claim 9, wherein the formulation or pesticide composition is in the form of a powder.

12. The formulation or pesticide composition of claim 10, wherein the formulation or pesticide composition is in the form of a wettable powder.

13. A formulation or pesticide composition as claimed in claim 11 wherein form I of the powder has a particle size D ₉₀ 5 μm to 80 μm.

14. The formulation or pesticide composition of claim 12, wherein the wettable powder has a particle size D of form I ₉₀ 5 μm to 80 μm.

15. Use of the crystalline form I of any one of claims 1 to 5, the crude drug or the parent drug of claim 6, or the formulation or the pesticide composition of any one of claims 7 to 14 for controlling cucumber downy mildew.

16. Use of the crystalline form I of any one of claims 1 to 5 as a primary or parent drug of claim 6, or of the formulation or pesticide composition of any one of claims 7 to 14 for the preparation of a cucumber downy mildew control formulation.

Use of an amorphous form of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt for controlling cucumber downy mildew.

Use of an amorphous form of 5-amino-2-methyl-6- (2, 3,4,5, 6-hydroxycyclohexyloxo) pyran-3-ylamino- α -iminoacetate salt for the preparation of a cucumber downy mildew control formulation.